CN1814760A - Rice antiviral related gene OsDR8 - Google Patents

Abstract

This invention relates to a separating clone and function verification of DNA segment of related gene OsDR8 of the anti-diseases of rice, which utilizes the technology of transforming genes based on the RNA interference principle to connect the part of DNA segments of the OsDR8 gene with a carrier capable of expressing double-chain RNA and shifts them into the anti-disease rice to suppress the expression of OsDR8 gene in it.

Description

Rice antiviral related gene OsDR 8

Technical field

The present invention relates to gene engineering technology field.Be specifically related to the separating clone and the functional verification of a rice antiviral related gene OsDR 8.The ability significant correlation of the function of OsDR8 gene and paddy rice opposing disease.

Background technology

Plant is subjected to the infringement of multiple pathogen in the process of growth.The phytopathy original of a great variety comprises virus, bacterium, fungi and nematode etc.The pathogen invaded plants causes two kinds of results: breeding in host plant of (1) pathogenic agent success causes relevant illness; (2) host plant produces disease resistance response, kills pathogen or stops its growth.Utilizing the disease resistance of resistant gene resource improvement plant, is the basic outlet that pre-disease prevention is protected environment simultaneously again.

The disease resistance response of plant is the complex process that polygene participates in regulation and control.The gene that participates in the plant disease-resistant reaction is divided into two classes: (1) disease-resistant gene claims R (resistance) gene and (2) disease-resistant related gene again.

According to the understanding of present people's enantiopathy gene function, the product of this genoid mainly is as acceptor, and direct or indirect and cause of disease protein-interacting starts plant intravital disease-resistant signal conducting path (Tang etc., 1996, Science 274:2060-2063; Baker etc., 1997, Science 276:726-733; Jia etc., 2000, EMBO is J.19:4004-4014; Dangl and Jones, 2001, Nature 411:826-833; Nimchuk etc., 2001, Curr.Opin.Plant Biol.4:288-294).The disease resistance response strong resistance of disease-resistant gene mediation is good genetic resources.But owing to following reason, make and utilize disease-resistant gene improvement plant resistance to environment stress to be restricted: the resource-constrained of (1) disease-resistant gene, disease-resistant gene as the important disease bacterial leaf-blight of opposing paddy rice known at present is less than 30, and the disease-resistant gene of resisting the important disease-rice blast of another paddy rice also has only about 40; (2) disease-resistant gene has cause of disease kind and cause of disease physiological strain specificity, disease-resistant being limited in scope; (3) because the quick sudden change of cause of disease, the effect of a disease-resistant gene has often just been lost after several years or more than ten years.

Disease-resistant related gene is meant that all participate in the gene of disease resistance response except that disease-resistant gene, and their coded product participates in disease-resistant signaling molecule in the synthetic plant materials, participates in the signal conduction or participates in defense response etc.The common feature of this genoid is that their expression amount of pathogeny evoked back raises or minimizing, therefore people can be according to the difference of pathogeny evoked front and back expression of gene amount plant identification disease-resistant related gene (Maleck etc. on a large scale, 2000, Nature Genet.26:403-410; Schenk etc., 2000, Proc.Natl.Acad.Sci.USA 97:11655-11660; Zhou etc., 2002, Science in China 45:449-467).At present, the understanding of people's enantiopathy genes involved is limited.According to existing report, most disease-resistant related genes are made the resistance capacity of time spent separately may be littler than disease-resistant gene.But according to following reason, they are to be worth the genetic resources of exploitation energetically: (1) because the product of most disease-resistant related genes does not need directly and pathogen interacts, this genoid is the genetic resources with durable resistance; (2) disease resistance response that participates in of most of disease-resistant related genes does not have the cause of disease specificity, so they are the genetic resourceses with resistance of wide spectrum; (3) aboundresources of this genoid.

The disease resistance response of plant can be divided into two big classes.Investigators have given different titles to this two big class disease resistance response for a long time, as vertical resistance and horizontal resistance (Van Der Plank etc., 1968, Disease Resistance in Plants, Academic, New Youk), quality resistance and quantity resistance (Ou etc., 1975, Phytopathology 65:1315-1316), complete resistance and partial resistance (Parlevliet, 1979, Annu.Rev.Phytopathol.1:203-222).Quality resistance (or vertical resistance or complete resistance) is the disease resistance response of disease-resistant gene mediation.Quantity resistance (or horizontal resistance or partial resistance) is by quantitative trait locus (quantitative trait locus, QTL) Tiao Kong disease resistance response, it is considered to not have the cause of disease specificity, and resistance lasting (Roumen, 1994, Rice Blast Disease, Zeigler etc. write, CAB International, Cambridge, UK, pp.245-265).At present, people are not clear to the gene essence of plant disease-resistant QTL.Therefore, though identified a large amount of disease-resistant QTL in the paddy rice, as resisting bacterial leaf-blight QTL (Li etc., 1999, Mol.Gen.Genet.261:58-63), blast resisting QTL (Wang etc., 1994, Genetics 136:1421-1434; Chen etc., 2003, Proc.Natl.Acad.Sci.USA100:2544-2549), anti-banded sclerotial blight QTL (Li etc., 1995, Theor.Appl.Genet.91:382-388) and viral diseases QTL (Albar etc., 1998, Theor.Appl.Genet.97:1145-1154) etc., but these resistances QTL is not performed well in the improvement of rice varieties disease resistance.

In recent years the chromosome position of discovering a lot of disease-resistant related genes is corresponding with disease-resistant QTL, and pointing out these disease-resistant related genes may be exactly corresponding QTL.Corresponding this phenomenon with disease-resistant QTL of the chromosome position of disease-resistant related gene all is observed in various plants, comprises paddy rice (Xiong etc., 2002, Chinese science 45:518-526; Ramalingam etc., 2003, Mol.Plant-Microbe Interact.16:14-24; Wen etc., 2003, Mol.Gen.Genomics 269:331-339; Chu etc., 2004, Mol.Gen.Genomics 271:111-120), wheat (Faris etc., 1999, Theor.Appl.Genet.98:219-225), beans (Geffroy etc., 2000, Mol.Plant-Microbe Interact.13:287-296) and potato (Trognitz etc., 2002, Mol.Plant-Microbe Interact.15:587-597).These results separate, clone and utilize the gene of disease-resistant QTL that foundation is provided for adopting candidate gene strategy.

Paddy rice is an important crops in the world, but the influence of disease usually causes the decline of its yield and quality.Therefore, understand the pathogenesis of disease, help to utilize the resistance of efficient approach improvement rice varieties, the generation of control disease, the loss that reduces or avoid Plant diseases to bring.

The separating clone disease-resistant related gene is the prerequisite to the research of paddy disease-resistant mechanism.Simultaneously, compare with the application of disease-resistant gene, the application of disease-resistant related gene can provide plant more wide spectrum and long lasting resistance.Carry out the improvement of rice varieties by the overexpression disease-resistant related gene, will further strengthen the disease resistance of plant, widen the anti-spectrum of plant.These aspects are that employing conventional plant breeding and improving technology institute are inaccessiable.

Summary of the invention

A disease-resistant related gene global DNA fragment that the objective of the invention is to carry in the separating clone paddy rice, and utilize RNA perturbation technique (RNA interference, RNAi) by making reticent this gene role in the disease resistance response process of identifying of target gene, for the ability of utilizing this improvement of genes rice varieties or other plant to resist disease lays the foundation.

The present invention relates to separate the dna fragmentation of a kind of OsDR8 of comprising (Oryza sativa defense responsive 8) gene and identify its function, this fragment is given plant to producing disease resistance response by bacterial leaf spot pathogenic bacteria (Xanthomonas oryzae pv.oryzae) and the caused disease of Pyricularia oryzae (Pyricularia grisea).Wherein, described fragment perhaps is equivalent to the dna sequence dna shown in the SEQ ID NO:1 basically shown in sequence table SEQ ID NO:1, and perhaps its function is equivalent to the subfragment of sequence shown in the SEQ ID NO:1.Its sequence is carried out the biosynthetic enzyme genes thil homology of the VITMAIN B1 of analysis revealed it and corn.

Can adopt the OsDR8 gene of having cloned to make probe, from cDNA and genomic library, screen gene of the present invention or homologous gene.Equally, adopt PCR (polymerase chain reaction) technology, also can from genome, mRNA and cDNA, increase obtain OsDR8 gene of the present invention and any interested section of DNA or with its homologous section of DNA.Adopt above technology, can separate the sequence that obtains comprising the OsDR8 gene, this sequence is connected with suitable carriers, can change vegetable cell over to, produce transgenic plant.

Change clone's disease-resistant related gene over to susceptible plant, help to produce new disease-resistant plants.Particularly can be with genetic transformation technology a plurality of resistant genes that in plant, add up, and can not produce the linked gene group sequence of following appearance in the traditional breeding technology.The clone of disease-resistant related gene overcomes traditional breeding method can not shift the disease-resistant related gene problem between plant species prerequisite.

In the embodiments of the invention part, we have set forth the separation of OsDR8 gene and the characteristics of functional verification process and this gene, isolating OsDR8 gene can be connected with appropriate carriers, changes in the plant materials, obtains or strengthen disease-resistant ability a possible approach is provided for making plant materials.

Sequence table, accompanying drawing and explanation thereof

The aminoacid sequence of the sequence of sequence table SEQ ID No:1.OsDR8 gene and its encoded protein matter.

Fig. 1. the schema of evaluation of the present invention and separating clone rice antiviral related gene OsDR 8 and checking OsDR8 gene function.

Fig. 2. be the queueing discipline figure that cDNA clones in each grid in the cDNA chip.384 grids are arranged on every nylon membrane of cDNA chip, and each grid has 8 sites.Show the arrangement mode of cDNA clone in the grid among the figure, each clone occupies two sites.A, B, C and D represent different cDNA clones respectively.

Fig. 3. utilize the cDNA clone of cDNA chip detection inoculation pathogenic bacteria front and back differential expression.A, c, e: with the probe and the chip hybridization of the leaf tissue making of inoculation pathogenic bacteria after 5 days; B, d, f: probe and the chip hybridization of using the leaf tissue making of water receiving (contrast).Grid on the box indicating cDNA chip has 8 sites (Fig. 2) in each grid.The cDNA of differential expression clone in the correlated image about arrow is illustrated in.3 cDNA shown in figure a, c and the e be cloned in the inoculation pathogenic bacteria after 5 days expression amount obviously increase.

Fig. 4 .OsDR8 gene is positioned at No. 7 karyomit(e)s of paddy rice, and its chromosome position and known blast resisting QTL (Wang etc., 1994, Genetics 136:1421-1434) are corresponding.

Fig. 5. adopt the expression characteristic of RT-PCR technical Analysis OsDR8 gene behind anti-rice blast rice material (C101LAC, C101A51 and bright extensive 63 (Minghui63)), sense rice blast rice material (CO39 and precious Shan 97 (Zhenshan 97)), resisting bacterial leaf-blight rice material (IRBB4, IRBB13 and bright extensive 63), sense bacterial leaf-blight rice material (IR24 and precious Shan 97) inoculation Pyricularia oryzae (IK81-3 and V86013), bacterial leaf spot pathogenic bacteria (PXO61, PXO99 and JL691) or water receiving (control, contrast).1,2,3,4,5,6,7 and 8 behind representative inoculation or the water receiving 2 hours, 4 hours, 8 hours, 16 hours, 1 day, 3 days, 5 day and 7 days respectively.With not being subjected to pathogeny evoked rice actin (actin) expression of gene amount as the standard of weighing sample size.

Fig. 6. cover the overlay chart of the subclone of OsDR8 constant gene segment C in the rice varieties bright extensive 63.The dna fragmentation that comprises the OsDR8 gene that the representative of figure middle or long line bar obtains from the bright extensive 63 BAC clone 5F21 of rice varieties, and the position in the digestion with restriction enzyme site in this bar segment.The position of arrow and length are represented length and the sequence location to restriction enzyme subclone order-checking, and the direction of arrow is represented direction that subclone is checked order.The splicing of subclone sequencing sequence is obtained the sequence of a long 1980bp.

Fig. 7. adopt GENSCAN (http://genes.mit.edu/GENSCAN.html) gene structure forecasting software, with reference to Arabidopis thaliana as analyze the 1980bp that template analysis obtains, the sequence that comprises the OsDR8 gene from rice varieties bright extensive 63.Analytical results shows that this sequence comprises the encoding sequence of OsDR8 gene complete.Gn represents the gene numbering among the figure; Ex represents exon; Init represents gene initiating terminal exon; Term represents gene clearing end exon; PlyA represents PolyA; S representation DNA chain, wherein the dna sequence dna chain of importing in "+" expression analytic process; Begin represents exon, promotor or the PolyA zero position in the input dna sequence dna; End represents exon, promotor or the PolyA final position in the input dna sequence dna; Len represents the sequence length (bp) of exon, promotor or PolyA; Frame (every dna sequence dna has 3 translations to read frame) is read in Fr representative translation; I/Ac represents 3 ' shearing site score value; Do/T represents 5 ' shearing site score value; CodRg representative translation distinguishing value; P represents the exon probability; Tscr represents the exon score value.

The structure of Fig. 8 .OsDR8 gene and be used for the dna fragmentation length and the structure of overexpression genetic transformation vector construction.Lines are represented intron; The post bar is represented exon, and wherein black is partly represented encoding sequence, and white portion is represented 5 ' and 3 ' non-coding region (UTR); The base number of each structure of numeral." ATG " and " TGA " is respectively translation initiation password and termination codon; " gt " and " ag " is the intron shearing site.Long arrow is represented the position of segmental length of genetic transformation and corresponding gene structure.Used 5 ' RACE and RT-PCR primer location during short arrow representative carrying out gene structure is analyzed.

Fig. 9. use quantitative RT-PCR technology for detection OsDR8 gene at control material (bright extensive 63) and T ₀For the expression amount in the genetic transformation plant (D16RMH).D16RMH-24 and D16RMH-30 are negative transformed plants, and other plant is positive transformed plant.Ordinate zou is represented the relative expression quantity (expression amount of control material be decided to be 1) (mean values of three experiments) of OsDR8 gene in every part of rice material.

Figure 10. spot-inoculated Pyricularia oryzae V86013 is at T ₀For easier growth and breeding on the genetic transformation plant (D16RMH), form obviously big, brown point.Arrow shows on the blade of part transformed plant and has formed scab.Bright extensive 63 is genetic transformation acceptor material (contrast), and Mudanjiang 8 is susceptible variety (contrasts).Four points of each blade inoculation, every bit inoculation bacterium liquid 10 microlitres.Inoculate back 4 days photos.

Embodiment

Further definition the present invention in following examples, Fig. 1 have described the flow process of evaluation and separating clone OsDR8 gene and checking OsDR8 gene function.According to above description and these embodiment, those skilled in the art can determine essential characteristic of the present invention, and under the situation that does not depart from spirit and scope of the invention, can make various changes and modification, so that its suitable various uses and condition to the present invention.

Embodiment one: make up pathogeny evoked rice cDNA library, make the rice cDNA chip

The rice at whole growth periods equilibration cDNA library (Chu Zhaohui etc. that adopt bright extensive 63 (the Oryza sativa ssp.indica) of disease resisting rice kind to make up, the structure of rice at whole growth periods normolized cDNA library and evaluation, 2002, Science Bulletin 47:1656-1662) making cDNA chip.This library is made up of the cDNA of 15 kinds of tissues under paddy rice different growing stage and the different physiological statuss, comprises the tissue after the inoculation of bacterial leaf spot pathogenic bacteria and Pyricularia oryzae is induced.This library comprises about 62,000 clones, and on average inserting fragment is 1.4kb.Get 21504 cDNA clones in this library at random, the extracting plasmid is made cDNA chip (cDNA array) (Zhou etc., The defense-responsive genes showing enhance and repressed expression after pathogeninfection in rice (Oryza sativa L.), 2002, Sci.in China 45:449-467).In order to guarantee the reliability of results of hybridization, each cDNA clone has two sites on the cDNA chip, and they are positioned at the symmetric position (Fig. 2) of same grid.

Embodiment two: utilize the disease-resistant related gene in the cDNA chip technology evaluation paddy rice

Inoculate the pathogenic bacteria (table 1) that is non-compatible host response with them respectively with four rice materials that carry bacterial leaf spot resistant ospc gene or blast resistant gene, with before the cause of disease inoculation and inoculate back 1 day with the total RNA of 5 days rice leaf respectively reverse transcription become cDNA and mark radio isotope to make probe, and respectively with the cDNA chip hybridization, the variation of the hybridization signal in the same cDNA site by analyzing different probe hybridization, identify the cDNA clone of a collection of cause of disease inoculation front and back differential expression, the different disease-resistant related gene (Fig. 3) of their representatives (referring to Zhou etc., The defense-responsive genes showing enhance andrepressed expression after pathogen infection in rice (Oryza sativa L.), 2002, Sci.in China45:449-467).

Table 1. is used for the disease resisting rice material and the pathogenic bacteria combination of cDNA chip analysis

Rice material	Disease-resistant gene	Non-affinity pathogenic bacteria
			IRBB10 IRBB13 C101A51 bright extensive 63	Bacterial leaf spot resistant ospc gene Xa10 bacterial leaf spot resistant ospc gene xa13 blast resistant gene Pi2 bacterial leaf spot resistant ospc gene Xa25 and Xa26, unnamed blast resistant gene	Bacterial leaf spot pathogenic bacteria PXO86 bacterial leaf spot pathogenic bacteria PXO99 Pyricularia oryzae V86013 Pyricularia oryzae V86013

Expression amount had increased by 3.2 times after the cDNA that wherein is numbered EI35I3 was cloned in the bright extensive 63 inoculation rice blast fungi isolates V86013 of rice varieties.In view of EI35I3 is cloned in pathogenic bacteria inoculation front and back expression amount notable difference is arranged, we think that the gene of cDNA clone EI35I3 representative has participated in the disease resistance response of plant, are disease-resistant related genes, and with its called after OsDR8.

Embodiment three: location OsDR8 gene on the molecule marker genetic linkage map

With one three hand over segregating population (Wang etc., 1998, Theor.Appl.Genet.97:407-412) the disease-resistant relevant cDNA clone EI35I3 that identifies is carried out the chromosomal localization analysis.Find that the OsDR8 gene is positioned at No. 7 karyomit(e)s of paddy rice, its chromosome position and known blast resisting QTL (Wang etc., 1994, Genetics 136:1421-1434)) corresponding (Fig. 4), prompting OsDR8 may be exactly the gene (Wen etc. of corresponding disease-resistant QTL, Three types of defense-responsivegenes are involved in resistance to bacterial blight and fungal blast diseases in rice, 2003, Mol.Gen.Genomics 269:331-339).

Embodiment four: the expression pattern analysis of OsDR8 gene in different paddy disease-resistant kinds

For further check analysis OsDR8 is a gene that rises through pathogen abduction delivering amount, the present invention at first checks order to cDNA clone EI35I3 and obtains the cDNA sequence (being positioned at 581 to 1817bp places of sequence shown in the sequence table SEQ ID NO:1) of a long 1159bp.Then according to cDNA sequences Design PCR primer, adopt RT-PCR (reverse transcriptase-PCR) technology, utilize 12 kinds of rice materials-pathogenic bacteria combinatory analysis OsDR8 expression of gene pattern (table 2) (Wen etc., Three typesof defense-responsive genes are involved in resistance to bacterial blight and fungal blast diseasesin rice, 2003, Mol.Gen.Genomics 269:331-339).In the analyzed rice material, C101A51, C101LAC and CO39 are the blast resisting near isogenic lines, and IRBB4, IRBB13 and IR24 are near isogenic line resisting bacterial leaf-blights.In RT-PCR analyzed, OsDR8 gene specific PCR primer 35I3F (5 '-TCCAGCCTCCTCAAGACCT-3 ') and 35I3R (5 '-AGTCCTCCTGCTTCGTCGTA-3 ') were according to the sequences Design of the cDNA fragment cloning EI35I3 of this gene; In addition, the standard of measuring as sample RNA with the amplified production of the PCR primer actinF (5 '-TATGGTCAAGGCTGGGTTCG-3 ') of rice actin (actin) and actinR (5 '-CCATGCTCGATGGGGTACTT-3 ').RT-PCR carries out (Zhou etc., 2002, Science in China 45:449-467) with two-step approach.At first, will be used for the DNA enzyme I processing of total RNA of reverse transcription, remove the DNA that may pollute among total RNA with no RNA enzymic activity; Total RNA was handled deactivation DNA enzyme I 10 minutes at 65 ℃; RNA 72 ℃ of sex change 5 minutes, is added Oligo-dT then ₁₅, dNTP, ThermoScript II etc., 42 ℃ of reverse transcriptions 90 minutes.In second step, get 1 microlitre reverse transcription product and make pcr amplification.

Table 2. is used for rice material and the pathogenic bacteria combination that RT-PCR analyzes

Rice material	Disease-resistant gene	Non-affinity pathogenic bacteria	The affinity pathogenic bacteria
				C101A51 CO39 C101LAC CO39 bright extensive 63	The susceptible unnamed blast resistant gene of the susceptible blast resistant gene Pil of blast resistant gene Pi2	Pyricularia oryzae V86013 Pyricularia oryzae IK81-3 Pyricularia oryzae V86013	Pyricularia oryzae V8601 Pyricularia oryzae IK81-3

Precious Shan 97 IRBB4 IR24 IRBB13 IR24 bright extensive 63 precious Shans 97

Xa26 is susceptible for the susceptible bacterial leaf spot resistant ospc gene of susceptible bacterial leaf spot resistant ospc gene Xa4 susceptible bacterial leaf spot resistant ospc gene xa13

Bacterial leaf spot pathogenic bacteria PXO61 bacterial leaf spot pathogenic bacteria PXO99 bacterial leaf spot pathogenic bacteria JL691

Pyricularia oryzae V86013 leaf spot bacteria PXO61 leaf spot bacteria PXO99 leaf spot bacteria JL691

After inoculating Pyricularia oryzae or bacterial leaf spot pathogenic bacteria as can be known from RT-PCR analytical results (Fig. 5), the expression amount of OsDR8 gene in all disease resisting rice materials all increased, and promptly pathogenic bacteria can be induced and strengthen OsDR8 genetic expression.This is induced and occurs in cause of disease the earliest and infect back 16 hours (Fig. 5).And the OsDR8 expression of gene is behind susceptible rice material inoculation pathogenic bacteria and the back no changes of all rice material water receivings (contrast).These results confirm that further OsDR8 is a disease-resistant related gene, and it not only participates in the regulation and control of rice bacterial blight resistance reaction, also participate in the regulation and control of blast resisting reaction.

Embodiment five: separating clone OsDR8 gene and gene structure analysis

1. with the prediction of OsDR8 dna homolog gene structure

Sequence with the cDNA fragment cloning EI35I3 of OsDR8 gene is made template, with BLAST analytical procedure (Altschul etc., 1997, Nucleic Acids Res.25:3389-3402) retrieval Nucleotide database, find in the database to be positioned at No. 7 karyomit(e)s of paddy rice from fine one of rice varieties Japan, (Nucleotide database GenBank (http://www.ncbi.nlm.nih.gov) number of registration: one section sequence AP004674) (the 23781st to 25016bp place) reaches 94% (E value=0) with the homology of EI35I3 to long 144741bp sequence, comprise and OsDR8 homologous gene, the i.e. allelotrope of OsDR8 among the prompting AP004674.

Utilize GENSCAN (http://genes.mit.edu/GENSCAN.html), GeneFinder (http://genomic.sanger.ac.uk/gf/gf.shtml), Gene Feature Searches (http://dot.imgen.bem.tmc.edu:9331/), the forecasting software of GeneMark a plurality of gene structures such as (http://genemark.biology.gatech.edu/GeneMark/hmmchoice.html), analyze in the AP004674 sequence section with EI35I3 cDNA sequence homology, infer in the AP004674 sequence coding region total length 1059bp with EI35I3 cDNA homologous gene.

2. gene terminal sequence analysis

Adopt 5 ' RACE test kit of American I nvitrogen company,, determine 5 ' end sequence of OsDR8 gene by 5 '-RACE (rapid amplification of cDNAend) analytical procedure.Carry out reverse transcription condition that 5 '-RACE analyzes and the reverse transcription term harmonization of the RT-PCR in the foregoing description four, the reverse transcription primer is OsDR8 gene specific primer GSP1 (5 '-GTAGGTGATCATGTCG-3 ').After reverse transcription is finished, carry out pcr amplification, amplimer is OsDR8 gene specific primer GSP2 (5 '-GGACTACCGCAACTCCA-3 ') and 5 ' RACE test kit anchor primer AbridgedAnchor (5 '-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3 ').Reclaim amplified fragments by electrophoresis then, clone reclaiming fragment with pGEM-T Vector System I test kit (U.S. Promega Corporation).At last the clone is carried out sequencing analysis.Sequential analysis finds that OsDR8 genetic transcription initiation site is positioned at the 349bp place, translation initiation site upstream of GENSCAN prediction, be 5 ' terminal non-translational region (un-translated region, UTR) the long 349bp (being positioned at 268 to 616bp places of sequence shown in the sequence table SEQ ID NO:1) of OsDR8 gene.

Clone the structure of the sequence homology gene of EI35I3 in the AP004674 sequence of the Nucleotide database GenBank that reference is inferred with cDNA, and compare, find that the EI35I3 sequence comprises 3 '-UTR of OsDR8 gene with the EI35I3 sequence.The long 64bp of 3 '-UTR (being positioned at 1754 to 1817bp places of sequence shown in the sequence table SEQ ID NO:1).Hence one can see that, and the ripe transcripton total length of OsDR8 gene is 1472bp (is positioned at 268 to 1817bp places of sequence shown in the sequence table SEQ ID NO:1, do not comprise intron).

3. separating clone OsDR8 gene from rice varieties bright extensive 63

Embodiment four has proved that rice varieties bright extensive 63 carries the OsDR8 gene of function.The present invention utilizes cDNA clone EI35I3 to make probe, screens bright extensive 63 BAC (bacterial artificial library) libraries (Peng etc., 1998, Botany Gazette 40:1108-1114), obtains a positive colony 5F21.Analyze in the AP004674 sequence digestion with restriction enzyme site with both sides, the homogenic coding region of EI35I3 cDNA, the digestion site of finding restriction enzyme SamI and BamHI lays respectively at the both sides with EI35I3 sequence homology gene coding region.Digest bright extensive 63 BAC clone 5F21 with SamI and BamHI, obtain the dna fragmentation of an about 1.9kb.With this dna fragmentation be connected to plasmid vector pCAMBIA1301 (see Sun etc., 2004, Plant is J.37:517-527; Wang Shi's equality, the Chinese invention patent ublic specification of application, number of patent application: 02139212.9), and name D55S.

Use restriction enzyme PstI, SacI, KpnI and BamHI+SacI digested plasmid D55S respectively.Reclaim endonuclease bamhi by 1% agarose gel electrophoresis, use T behind the purifying ₄-archaeal dna polymerase is mended flat terminal, be connected with dephosphorylized pUC19 carrier (the U.S. Amersham Bioscience company) flush end of restriction endonuclease sma I digestion process, electricity transformed into escherichia coli DH10B (Sun etc., 2004, Plant is J.37:517-527), obtain positive subclone by blue hickie screening.Extract positive subclone plasmid, and carry out electrophoresis relatively, reject false positive clone and cloning of small fragment, detect positive colony and insert clip size with empty pUC19 plasmid.

Adopt the sequencing kit (Big Dye Kit) of M13-R and M13-F universal primer, U.S. Perkin Elmer company, with the terminal cessation method of dideoxy nucleotide (U.S. Perkin Elmer company) respectively from the one or both ends order-checking of each subclone.Altogether 6 subclones have been carried out check order (Fig. 6).Sequence to 6 subclones is spliced, and obtains the sequence of a long 1980bp.Analyze the long sequence of this 1980bp, prove that it comprises the complete encoding sequence of OsDR8 gene.

4.OsDR8 determining of gene intron

Adopt predictive genes software GenScan (http://genes.mit.edu/GENSCAN.html) that the analysis of the bright extensive 63 genomic 1980bp dna sequence dnas that comprise the OsDR8 gene is shown in its coding section and have an intron (Fig. 7).The present invention compares according to cDNA clone's EI35I3 sequencing result (being positioned at 581 to 1817bp places of sequence shown in the sequence table SEQ ID NO:1) and clone D55S sequencing result, determines to exist really in 1502 to 1579bp sequences of sequence shown in the sequence table SEQ ID NO:1 intron of a 78bp.The size of the intron of this intron and prediction and position consistency (Fig. 7).

In order to determine whether the OsDR8 gene exists other intron, the cDNA sequence of EI35I3 is spliced the back with the cDNA sequence (being positioned at 268 to 784bp places of sequence shown in the sequence table SEQ ID NO:1) of carrying out 5 ' RACE analysis acquisition to be analyzed with clone D55S sequence, determine that the OsDR8 gene only comprises the intron of a 78bp, it is positioned at 1502 to 1579bp places of sequence shown in the sequence table SEQ ID NO:1.According to this as a result the OsDR8 gene coding region be divided into two sections by intron, long respectively 885bp (being positioned at 617 to 1501bp places of sequence shown in the sequence table SEQ IDNO:1) and 174bp (being positioned at 1580 to 1753bp places of sequence shown in the sequence table SEQ ID NO:1) are (Fig. 8).OsDR8 full length gene 1550bp (being positioned at 268 to 1817bp places of sequence shown in the sequence table SEQ ID NO:1) (Fig. 8).

Embodiment six: the analysis of OsDR8 gene encoding production

According to the BLAST analytical results, the protein of OsDR8 genes encoding and plant vitamin B1 synthetic enzyme have higher homology.Coded product (National Center for Biotechnology Information (NCBI as the biosynthetic enzyme genes thil-1 of the VITMAIN B1 of the coded product of OsDR8 gene and corn, http://www.ncbi.nlm.nih.gov) Protein Data Bank number of registration: S61419) homology, (E value=e-142) that the amino acid consistence is 73%, coded product (NCBI Protein Data Bank number of registration: S61420) homology with the biosynthetic enzyme genes thil-2 of the VITMAIN B1 of corn, the amino acid consistence is 73%, and (E value=e-141) is with the biosynthetic enzyme of the VITMAIN B1 of sweet orange (NCBI Protein Data Bank number of registration: T10474) amino acid consistence (the E value=e-132) that is 72%.Above evidence explanation, the coded product of OsDR8 gene very likely is a kind of VITMAIN B1 synthetic enzyme.

Embodiment seven: the functional verification of OsDR8 gene

The present invention adopt RNA disturb (RNA interference, RNAi) technology, by suppressing OsDR8 expression of gene in the disease resisting rice kind bright extensive 63, verify the function of this gene.The dominant mechanism of this technological approaches: (double strand RNA, carrier dsRNA) connects, and by genetic transformation this carrier is imported in plant with expressing double-stranded RNA with reverse multiple form with the part fragment of target gene.The transformed plant great expression and the target gene part fragment homologous dsRNA that obtain.These dsRNA form rapidly short interfering rna (short interfering RNA, siRNA).The transcripton of these siRNA and target gene (mRNA) complementary pairing makes the transcripton degraded of target gene under the effect of specific enzymes in cell, thereby suppress the function of target gene on the mRNA level.The investigator can be by the change of transfer-gen plant phenotype, the function of checking target gene.The RNA perturbation technique has been widely used in the checking (Smith etc., 2000, Nature 407:319-320) of gene function

The present invention with following dsF and dsR sequence as the PCR primer, cDNA clone (the EI35I3 that contains the OsDR8 gene cDNA fragment, (this carrier is seen the clear sunshine of storage etc. to the pSPORT1 carrier, the structure of rice at whole growth periods normolized cDNA library and evaluation, 2002, Science Bulletin 47:1656-1662)) be template, obtain dna fragmentation to be transformed by pcr amplification.Clone for the ease of the PCR product, the part that 5 ' of dsF and dsR primer-end indicates underscore is the digestion site of Restriction Enzyme SpeI and AscI or SacI and AvrII, the sequence of 3 '-end subsequently then respectively with the sequences match of carrier pSPORT1 multiple clone site both sides.

SpeI AscI

dsF：5’-TA ACTAGT GGCGCC TGCAGGTACCGGTCCG-3’

SacI AvrII

dsR：5’-TA GAGCTC GCCTAG GTGCACGCGTACGTACGTAAGC-3’

Make up first of OsDR8 gene fragment and repeat chain (forward chain): use AscI and AvrII digestion part PCR product and carrier pMCG161 (according to the operation instruction of carrier pMCG161, http://www.chromdb.org/info/plasmids/pMCG161.html), the complete back of digestion is at 75 ℃ of water-bath 10min deactivation restriction enzymes, place on ice, use T ₄Dna ligase carries out ligation.Electricity changes cloned plasmids dsF and the dsR primer amplification screening positive clone that the back obtains.

Make up second of OsDR8 gene fragment and repeat chain (reverse strand): digest remaining PCR product and the cloned plasmids that has been connected the first repetition chain with SacI and SpeI, digestion back is fully used T at 75 ℃ of water-bath 10min deactivation restriction enzymes ₄The D16A ligase enzyme carries out ligation.Electricity changes cloned plasmids SacI and the two enzymic digestion reaction of the SpeI screening positive clone that the back obtains, positive colony called after D16R.

The double-stranded RNA carrier D16R electricity that builds is transformed Agrobacterium competent cell EHA105, preserve bacterial strain and transform as agriculture bacillus mediated rice genetic.

Adopt agriculture bacillus mediated genetic transforming method (Lin and Zhang, Optimising the tissue culture conditions forhigh efficiency transformation of indica rice, 2005, Plant Cell Rep.23:540-547) D16R is imported disease resisting rice kind bright extensive 63.The genetic transformation plant that obtains is named as D16RMH (wherein D16R is the genetic transformation container name, and MH represents rice varieties bright extensive 63).The present invention obtains independent transformed plant 30 strains altogether.Whole 30 strain plant are become stage strain phase inoculation bacterial leaf-blight bacterial strain PXO61, finding the resistance weakened at different degrees of 15 strain transformed plants; Compare with D16RMH-30 with genetically modified acceptor rice varieties bright extensive 63 and negative transformed plant D16RMH-24, (7%-42% (table 3) is grown * %) increased to scab length/sick leaf to the lesion area of the transformed plant that these resistances weaken.

Table 3. part T ₀For the reaction of transformed plant (D16RMH) to bacterial leaf spot pathogenic bacteria strain PXO61

Rice material	Lesion area (%) (1)	The P value
			Bright extensive 63 D16RMH-1 D16RMH-2 D16RMH-3 D16RMH-5 D16RMH-8 D16RMH-9 D16RMH-11 D16RMH-12 D16RMH-13 D16RMH-15 D16RMH-16 D16RMH-17 D16RMH-21 D16RMH-23 D16RMH-29 D16RMH-24(2) D16RMH-30 (2)	17％ 39％ 57％ 60％ 47％ 23％ 37％ 55％ 50％ 35％ 59％ 48％ 44％ 47％ 35％ 37％ 17％ 17％	0.079 inoculation a slice leaf; Can't analyze 0.022 0.000 inoculation a slice leaf; Can't analyze 0.113 0.034 0.022 0.031 0.113 0.000 inoculation a slice leaf; Can't analyze inoculation a slice leaf, can't analyze 0.168 0.032 0.926 0.954

⁽¹⁾Every strain transfer-gen plant inoculation 3-5 sheet leaf except that the plant of special mark, two all " Invest, Then Investigate " scabs and sick leaf length, each data comes from the mean value of a plurality of blades.

⁽²⁾Negative transformed plant, the positive transformed plant of other plant.

For whether the further resistance against diseases of checking transformed plant weakens relevantly with the OsDR8 gene expression amount, listed 17 strain transformed plant extracted total RNA are utilized Rotor-Gene 3000 in the his-and-hers watches 3 of the present invention ^TMThe quantitative PCR analysis method of quantitative PCR instrument (Rotor-Gene company), SYBR Green fluorescence intercalative dye and Rotor-Gene company is done the quantitative RT-PCR analysis, relatively OsDR8 gene expression amount in transformed plant and the control material.PCR reaction primer is 35I3F (5 '-TCCAGCCTCCTCAAGACCT-3 ') and 35I3R (5 '-AGTCCTCCTGCTTCGTCGTA-3 ').RT-PCR product with rice actin contrasts as the sample size consistence.Actin muscle PCR primer sequence is actin (5 '-TGCTATGTACGTCGCCATCCAG-3 ') and actinR (5 '-AATGAGTAACCACGCTCCGTCA-3 ').

The phenotype of OsDR8 expression of gene situation and plant is divided into from (Fig. 9) in the experimental result demonstration transformed plant.The transformed plant that OsDR8 gene expression amount and disease resistance weaken is negative correlation; OsDR8 expression of gene amount is compared remarkable minimizing with control material bright extensive 63 in the transformed plant that disease resistance weakens.And disease-resistant phenotype does not have in the transformed plant (D16RMH-24 and D16RMH-30) of considerable change the OsDR8 gene expression amount and compares no considerable change with bright extensive 63.This presentation of results the OsDR8 gene in the middle of the rice bacterial blight resistance reaction process, bring into play considerable effect.

In addition, the present invention proves that also transformed plant has also weakened the resistance of Pyricularia oryzae.Get the blade of transformed plant and control material, adopt some inoculation (spot inoculation) method (Jia etc., Determination of host responses to Magnaporthegrisea on detached rice leaves using a spot inoculation method, 2003, Plant Disease 87:129-133) observes at indoor inoculation Pyricularia oryzae V86013, the growth and breeding situation of Pyricularia oryzae is all bright extensive 63 significantly better than the contrast disease-resistant variety on the transformed plant blade that the OsDR8 gene expression amount reduces, and similar to No. 8 situations in susceptible variety Mudanjiang; On the blade of part transfer-gen plant even formed typical scab (Figure 10).Illustrate that the OsDR8 gene also plays a role in the rice anti-rice blast reaction.

The biosynthetic enzyme genes thil height homology of the VITMAIN B1 of OsDR8 gene and corn.The protein of thil gene shows that in intracellular location situation it is on the plastid film, because the aminoterminal of the protein of this gene has a plastid positioning sequence, and a lot of hydrophobic amino acids is arranged, therefore infer that this place, gene product place plastid is the synthetic place (Belanger etc. of VITMAIN B1,1995, Plant Mol Biol, 29:809-821).VITMAIN B1 is the cofactor of metabolism related enzymes many in the organism, and these pathways metabolisms comprise tricarboxylic acid cycle, pentose-phosphate pathway, anaerobic respiration, the synthetic branch's approach of amino acid and pigment biosynthesizing etc.As the synthetic pathways metabolism that is experienced of β-Hu Luobusu pyruvic acid being converted into the required enzyme pyruvic oxidase of acetyl-CoA, to need VITMAIN B1 be cofactor, and being thiaminpyrophosphate, the VITMAIN B1 activity form participates in metabolism (Schulze-Siebert etc. as cofactor, 1987, Plant Physiol, 84:1233-1237).There are some researches show VITMAIN B1 except in the biochemical metabolism approach of some plants, working, it equally also can be used as the accumulation of an intravital pathogenesis-related proteins of inducible factor stimulating plant (PR-1), increase the resistance (Malamy etc. of tobacco to tobacco mosaic virus (TMV), 1996, Mol Plant-Microbe Interact, 9:474-482).In conjunction with this experimental result, infer that the function of OsDR8 gene may be after disease-resistant plants is subjected to pathogen infection, to induce the protein that has produced this gene, thereby promoted a large amount of synthetic of VITMAIN B1; And VITMAIN B1 can be converted into its activity form strengthens the relevant enzyme of some metabolism as cofactor activity, make plant produce a large amount of Physiology and biochemistries and change and resist disease, can induce the generation of pathogenesis-related proteins and control disease owing to the accumulation of VITMAIN B1 simultaneously.

Sequence table SEQ ID NO:1

＜110〉Hua Zhong Agriculture University

＜120〉rice antiviral related gene OsDR 8

<130>

<141>2005-01-30

<160>3

<170>PatentIn version 3.1

<210>1

<211>1980

<212>DNA

<213>Oryza sativa

<220>

<221>gene

<222>(268)..(1817)

<223>

<220>

<221>3’UTR

<222>(1754)..(1817)

<223>

<220>

<221>CDS

<222>(617)..(1501)

<223>

<220>

<221>CDS

<222>(1580)..(1753)

<223>

<220>

<221>Intron

<222>(1502)..(1579)

<223>

<220>

<221>5’UTR

<222>(268)..(616)

<223>

<400>1

gggctccacc actagtaccc ctcactacag gtagccataa aaaaaatcga tcaccaaaac 60

ccattattag gttgtgtact gatacagaaa gttgggaacc aatctcccag cacagaaaac 120

ggtacggttc attagcgcgt gattaattaa atatttacta ttttttaaaa aaaatagatc 180

aatatgattt ttaagcaact ttcgtataaa tactttttca aaaaaacaca ccgttttcta 240

gtttgaaaag cgtacacgcg tgaaatgagg gagaaaggtt ggaaacgtgg gattgcaaac 300

acagcattag tcgtcacggt acccagcaaa aaatcactga acacagcacc actgttcccg 360

tgagtccgta acttagcagc acttgtcccg ttctcctgaa gacagtccaa aaaccctcac 420

taaaccttcc caaaatatct cctccaatca atccgaaacc cagaggccca tactgctgac 480

acgtggacgg cactcccaga tatttgcgct ctcctcttct tcttataccc ccgccaaccc 540

acgctctatc cactcacaca cacactcctc atcccagagc aagaagctca gctcctcctc 600

ctctcgcatg gcagcc atg gcc acc acc gcg tcc agc ctc ctc aag acc tcc 652

Met Ala Thr Thr Ala Ser Ser Leu Leu Lys Thr Ser

1 5 10

ttc gcc ggc gtg cgc ctc ccc gcc gcc gcc cgc aac ccc acc gtc tcc 700

Phe Ala Gly Val Arg Leu Pro Ala Ala Ala Arg Asn Pro Thr Val Ser

15 20 25

gtc gcg ccg cgc acc gga ggc gcc atc tgc aac tcc atc tcg tcg tcg 748

Val Ala Pro Arg Thr Gly Gly Ala Ile Cys Asn Ser Ile Ser Ser Ser

30 35 40

tcg tcc act ccc ccc tac gac ctc aac gcc atc agg ttc agc ccc atc 796

Ser Ser Thr Pro Pro Tyr Asp Leu Asn Ala Ile Arg Phe Ser Pro Ile

45 50 55 60

aag gag tcc atc gtg tcc cgc gag atg acc cgg cgg tac atg acc gac 844

Lys Glu Ser Ile Val Ser Arg Glu Met Thr Arg Arg Tyr Met Thr Asp

65 70 75

atg atc acc tac gcc gac acc gac gtc gtc gtc gtc ggc gcc ggc tcc 892

Met Ile Thr Tyr Ala Asp Thr Asp Val Val Val Val Gly Ala Gly Ser

80 85 90

gcg ggg ctc tcc tgc gcg tac gag ctc tcc aag gac ccc tcc gtc agc 940

Ala Gly Leu Ser Cys Ala Tyr Glu Leu Ser Lys Asp Pro Ser Val Ser

95 100 105

gtc gcc gtc atc gag cag tcg gtg tcc ccc ggc ggc ggc gcg tgg ctc 988

Val Ala Val Ile Glu Gln Ser Val Ser Pro Gly Gly Gly Ala Trp Leu

110 115 120

ggc ggg cag ctg ttc tcc gcc atg gtg gtg cgc aag ccg gcg cac ctg 1036

Gly Gly Gln Leu Phe Ser Ala Met Val Val Arg Lys Pro Ala His Leu

125 130 135 140

ttc ctc gac gag ctc ggc gtc gcg tac gac gag cag gag gac tac gtc 1084

Phe Leu Asp Glu Leu Gly Val Ala Tyr Asp Glu Gln Glu Asp Tyr Val

145 150 155

gtc atc aag cac gcc gcg ctc ttc acc tcc acc gtc atg agc cgc ctc 1132

Val Ile Lys His Ala Ala Leu Phe Thr Ser Thr Val Met Ser Arg Leu

160 165 170

ctg gcg cgc ccc aac gtg aag ctg ttc aac gcc gtc gcc gtc gag gac 1180

Leu Ala Arg Pro Asn Val Lys Leu Phe Asn Ala Val Ala Val Glu Asp

175 180 185

ctc atc gtc aag aag ggc cgg gtc ggc cgg gtg gtc acc aac ttg ggc 1228

Leu Ile Val Lys Lys Gly Arg Val Gly Arg Val Val Thr Asn Leu Gly

190 195 200

gct tgt gtc aat gac cac gac acg cag ccg ggc atg gac cca aac gtg 1276

Ala Cys Val Asn Asp His Asp Thr Gln Pro Gly Met Asp Pro Asn Val

205 210 215 220

atg gag ttc aag gtg gtg gtg agc tcc tgc ggc cac gac ggg ccg ttc 1324

Met Glu Phe Lys Val Val Val Ser Ser Cys Gly His Asp Gly Pro Phe

225 230 235

ggc gcc acg ggc gtc aag cgg ctg cag gac atc ggc atg atc gac gcc 1372

Gly Ala Thr Gly Val Lys Arg Leu Gln Asp Ile Gly Met Ile Asp Ala

240 245 250

gtg ccc ggc atg cgc gcc ctc gac atg aac acc gcc gag gac gag atc 1420

Val Pro Gly Met Arg Ala Leu Asp Met Asn Thr Ala Glu Asp Glu Ile

255 260 265

gtc cgc ctc acc cgc gag gtc gtc ccc ggc atg atc gtc acc ggc atg 1468

Val Arg Leu Thr Arg Glu Val Val Pro Gly Met Ile Val Thr Gly Met

270 275 280

gag gtc gcc gag atc gac ggc gcc ccg aga atg gtacgcacca aaaaaacatc 1521

Glu Val Ala Glu Ile Asp Gly Ala Pro Arg Met

285 290 295

aaactcttca ccataatctc tcccccgttc ttggatttga acaaattcgt tgttgcag 1579

ggc ccg acg ttc gga gcc atg atg atc tcc ggc cag aag gcg gcg cac 1627

Gly Pro Thr Phe Gly Ala Met Met Ile Ser Gly Gln Lys Ala Ala His

300 305 310

ctg gcg ctg aag gcg ctc ggc cgg ccg aac gcc atc gac ggc acg atc 1675

Leu Ala Leu Lys Ala Leu Gly Arg Pro Asn Ala Ile Asp Gly Thr Ile

315 320 325

aag aag gcg gcg gcg gcg gcg gcg cac ccg gag ctg atc ctg gcg tcg 1723

Lys Lys Ala Ala Ala Ala Ala Ala His Pro Glu Leu Ile Leu Ala Ser

330 335 340

aag gac gac ggc gag atc gtg gac gcc tga gcaaataaaa cagggtaaaa 1773

Lys Asp Asp Gly Glu Ile Val Asp Ala

345 350

aaaaatcccc caaacgtggt ggtgacacgg aggggttggg gaccaaaaaa aaatgtggac 1833

tttcccctgt gttttttttt cgggatttgc tttgatcccc ttgtttgttt tagctttgga 1893

ggttgataag ggttttgtta aaaacaattc ccctcccacc gggtgtgtgt ggtttgcttg 1953

cctaatgagg gcaaaaaaac ttccatg 1980

<210>2

<211>295

<212>PRT

<213>Oryza sativa

<400>2

Met Ala Thr Thr Ala Ser Ser Leu Leu Lys Thr Ser Phe Ala Gly Val

1 5 10 15

Arg Leu Pro Ala Ala Ala Arg Asn Pro Thr Val Ser Val Ala Pro Arg

20 25 30

Thr Gly Gly Ala Ile Cys Asn Ser Ile Ser Ser Ser Ser Ser Thr Pro

35 40 45

Pro Tyr Asp Leu Asn Ala Ile Arg Phe Ser Pro Ile Lys Glu Ser Ile

50 55 60

Val Ser Arg Glu Met Thr Arg Arg Tyr Met Thr Asp Met Ile Thr Tyr

65 70 75 80

Ala Asp Thr Asp Val Val Val Val Gly Ala Gly Ser Ala Gly Leu Ser

85 90 95

Cys Ala Tyr Glu Leu Ser Lys Asp Pro Ser Val Ser Val Ala Val Ile

100 105 110

Glu Gln Ser Val Ser Pro Gly Gly Gly Ala Trp Leu Gly Gly Gln Leu

115 120 125

Phe Ser Ala Met Val Val Arg Lys Pro Ala His Leu Phe Leu Asp Glu

130 135 140

Leu Gly Val Ala Tyr Asp Glu Gln Glu Asp Tyr Val Val Ile Lys His

145 150 155 160

Ala Ala Leu Phe Thr Ser Thr Val Met Ser Arg Leu Leu Ala Arg Pro

165 170 175

Asn Val Lys Leu Phe Asn Ala Val Ala Val Glu Asp Leu Ile Val Lys

180 185 190

Lys Gly Arg Val Gly Arg Val Val Thr Asn Leu Gly Ala Cys Val Asn

195 200 205

Asp His Asp Thr Gln Pro Gly Met Asp Pro Asn Val Met Glu Phe Lys

210 215 220

Val Val Val Ser Ser Cys Gly His Asp Gly Pro Phe Gly Ala Thr Gly

225 230 235 240

Val Lys Arg Leu Gln Asp Ile Gly Met Ile Asp Ala Val Pro Gly Met

245 250 255

Arg Ala Leu Asp Met Asn Thr Ala Glu Asp Glu Ile Val Arg Leu Thr

260 265 270

Arg Glu Val Val Pro Gly Met Ile Val Thr Gly Met Glu Val Ala Glu

275 280 285

Ile Asp Gly Ala Pro Arg Met

290 295

<210>3

<211>57

<212>PRT

<213>Oryza sativa

<400>3

Gly Pro Thr Phe Gly Ala Met Met Ile Ser Gly Gln Lys Ala Ala His

1 5 10 15

Leu Ala Leu Lys Ala Leu Gly Arg Pro Asn Ala Ile Asp Gly Thr Ile

20 25 30

Lys Lys Ala Ala Ala Ala Ala Ala His Pro Glu Leu Ile Leu Ala Ser

35 40 45

Lys Asp Asp Gly Glu Ile Val Asp Ala

50 55

Claims (3)

1, give the dna sequence dna of paddy rice to bacterial leaf-blight and rice blast generation resistance, it is the dna sequence dna shown in the SEQ ID NO:1, and it comprises complete OsDR8 gene.

2, the coding region of OsDR8 gene, it is 617-1501 position and the dna sequence dna shown in the 1580-1753 position or coding and the identical protein DNA sequence of SEQ ID NO:1 encoded protein matter among the SEQ ID NO:1.

3, each described dna sequence dna of claim 1-2 is increasing paddy rice to the application in bacterial leaf-blight and the rice blast resistance.

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